Methods of preserving a biological sample

ABSTRACT

Provided herein are methods of preserving a biological sample on a first substrate, the methods including: (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, wherein the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the chamber; and (d) assembling the first substrate with a second substrate, thereby preserving the biological sample on the first substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/228,432, filed on Aug. 2, 2021, the contents of which are hereby incorporated by reference in its entirety.

BACKGROUND

Cells within a tissue have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, signaling, and cross-talk with other cells in the tissue.

Spatial heterogeneity has been previously studied using techniques that typically provide data for a handful of analytes in the context of intact tissue or a portion of a tissue (e.g., tissue section), or provide significant analyte data from individual, single cells, but fails to provide information regarding the position of the single cells from the originating biological sample (e.g., tissue).

It is desirable to preserve a biological sample to maintain the integrity (e.g., morphological characteristics) of the biological sample (e.g., tissue) for present and future analysis, including spatial transcriptomic analysis. However, detachment of a biological sample disposed on a substrate can occur when performing various analysis methods, including spatial analysis methods. The present disclosure features methods, compositions, and kits for preserving a biological sample on a substrate and for limiting or preventing detachment of the biological sample.

SUMMARY

Biological samples can be preserved on a substrate (e.g., a slide) for further analysis (e.g., microscopy, spatial analysis, and/or immunohistochemistry) to maintain the integrity (e.g., the physical characteristics) of the tissue structure. Described herein are methods of preserving a biological sample on a first substrate (e.g., glass slide) using a composition including a mixture of (i) a hydrophobic agent (e.g., grease, polyethylene glycol, etc.) and (ii) a plurality of beads, where the composition provides a customizable spacer agent between the first substrate and a second substrate (e.g., a cover slide).

In some examples, a biological sample can be preserved between a first substrate and a second substrate, where the composition is applied around the biological sample, thereby creating a chamber where the biological sample is preserved. In some examples, the gap distance between the first and second substrate is about the diameter of the beads. In some examples a mounting agent (e.g., a saccharide) can be placed in the chamber (e.g., in combination with the biological sample). In some examples, the composition can be removed from the first substrate before analysis of the biological sample by applying heat. Application of heat results in partial or complete melting of the composition such that the composition can be removed from the substrate. Preserving the biological sample with a composition including a mixture of a hydrophobic agent and a plurality beads and a mounting agent can help prevent sample detachment when a biological sample is disposed between two substrates, thereby preserving it for future use and analysis. Thus, in some examples, the various methods, compositions, and kits of preserving biological samples on a substrate (e.g., a first substrate) as described herein can prevent sample detachment from the substrate.

Provided herein are methods of preserving a biological sample on a first substrate, the method including: (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, where the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the chamber; and (d) assembling the first substrate with a second substrate, thereby preserving the biological sample on the first substrate.

In some embodiments, the first substrate and/or the second substrate includes a glass slide.

In some embodiments, the first substrate includes a plurality of capture probes, where a capture probe of the plurality of capture probes includes (i) a spatial barcode and (ii) a capture domain.

In some embodiments, the second substrate includes a porous surface, where the porous surface includes a cellulose membrane.

In some embodiments, the method includes fixing the biological sample after the first substrate and the second substrate are assembled with the biological sample. In some embodiments, the biological sample is fixed with formaldehyde, methanol, or acetone.

In some embodiments, the spacer agent includes a mixture including a blocking paste and a plurality of beads. In some embodiments, the mixture includes from about 1×10⁸ beads/mL in about 0.5 cm³ of the blocking paste to about 1×10¹² beads/mL in about 2 cm³ of the blocking paste.

In some embodiments, the blocking paste includes a hydrophobic agent. In some embodiments, the hydrophobic agent includes a grease, where the grease includes one or more of a vacuum grease, an apiezon grease, a lithium grease, a silicone grease, a Fomblin grease, and a TLC grease.

In some embodiments, the method includes partially or completely melting the grease and removing the second substrate from the first substrate.

In some embodiments, the grease has a melting temperature of about 25° C. to about 250° C. In some embodiments, the hydrophobic agent includes polyethylene glycol (PEG), where the PEG has a molecular weight of about 400 daltons to about 6,000 daltons, and where the PEG has a melting temperature between about 25° C. and 250° C.

In some embodiments, a bead in the plurality of beads has a diameter from about 5 μm to about 80 μm. In some embodiments, the spacer agent generates a gap distance between the first substrate and the second substrate, where the gap distance is about the diameter of the bead.

In some embodiments, the mounting agent includes glycerin or a saccharide.

In some embodiments, the biological sample includes a tissue sample, a tissue section, an organ, an organism, or a cell culture sample.

In some embodiments, the method includes imaging the biological sample.

In some embodiments, the method includes: (i) separating the first substrate from the second substrate; (ii) contacting the first substrate with an array including a plurality of capture probes, where a capture probe of the plurality capture probes includes: (i) a spatial barcode and (ii) a capture domain; and (iii) performing spatial transcriptomic analysis.

In some embodiments, the spatial transcriptomic analysis includes sequencing.

Also provided herein are methods of preserving and analyzing a biological sample, the method including: (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, where the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the biological sample within the chamber; (d) analyzing the biological sample on the first substrate; (e) assembling the first substrate with a second substrate such that the chamber is sealed, thereby preserving the biological sample on the first substrate; (f) applying heat to the first substrate, thereby partially or completely melting the spacer agent and removing the second substrate and the spacer agent from the first substrate; (g) contacting the first substrate with an array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; and (h) performing spatial transcriptomic analysis.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

The term “about” or “approximately” as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

FIG. 1A shows an exemplary configuration of a first substrate, a spacer agent, a mounting agent, and a second substrate.

FIG. 1B shows a side view of the exemplary configuration in FIG. 1A of a first substrate, a spacer agent, and a second substrate.

FIG. 2A shows an exemplary image of different spacer agents applied to glass slides in different configurations (e.g., rectangular, square, and triangle).

FIG. 2B shows an exemplary image of the different spacer agents applied in different configurations on the glass slide, including a mounting agent deposited to the chamber internal to the spacer agent configuration.

FIG. 3 shows an exemplary image of a spacer agent applied to a first substrate in a rectangular configuration, a mounting agent delivered to the chamber, and a second substrate assembled on top of the first substrate and parallel to the first substrate, wherein the chamber is completely sealed and the gap between the first and second substrate is defined by the diameter of the plurality of beads in the spacer agent (e.g., about 10 μm).

FIG. 4 shows an exemplary image of a spacer agent applied to a first substrate in a triangular configuration, a mounting agent delivered to the chamber, and a second substrate assembled with the first substrate in a perpendicular manner, wherein the chamber is completely sealed and the gap between the first and second substrate is defined by the diameter of the plurality of beads in the spacer agent (e.g., about 20 μm).

FIG. 5 shows an exemplary image of a spacer agent applied to the first substrate in a square configuration, a mounting agent delivered to the chamber, and a second substrate assembled with the first substrate in a perpendicular manner, wherein the chamber is completely sealed and the gap between the first and second substrate is defined by the diameter of the plurality of beads in the spacer agent (e.g., about 30 μm).

FIG. 6 shows an exemplary image of a spacer agent applied to the first substrate in a triangular configuration, a mounting agent delivered to the chamber, and a second substrate assembled with the first substrate in a perpendicular manner, wherein the chamber is completely sealed and the gap between the first and second substrate is defined by the diameter of the plurality of beads in the spacer agent (e.g., about 40 μm).

FIG. 7 shows an exemplary image wherein the second substrate is removed by gently melting the spacer agent, wherein a biological sample preserved in the chamber can be recovered for further analysis.

DETAILED DESCRIPTION

Biological samples can be prepared and preserved on a substrate (e.g., a slide, an array) to maintain the integrity (e.g., the physical characteristics) of the tissue structure for sample analysis (e.g., microscopy, spatial profiling, tissue staining and/or immunohistochemistry, etc.) to be performed at a later time. Described herein are methods of preserving a biological sample on a first substrate by using a composition including a mixture of (i) a hydrophobic agent (e.g., grease, polyethylene glycol (PEG), etc.) and (ii) a plurality of beads, where the composition provides a customizable gap between the first substrate and a second substrate (e.g., a cover slide). As used herein, “customizable” refers to the gap between the first substrate and second substrate established by the diameter of the plurality of beads. For example, the diameter of the plurality of beads establishes the gap distance or height between the first substrate and second substrate and thus, beads with different diameters allow one of ordinary skill in the art to control (e.g., customize) the gap distance.

In some examples, a biological sample can be preserved between a first substrate and a second substrate, wherein the composition is applied around the biological sample, thereby creating a chamber where the biological sample is preserved. In some examples, the gap distance between the first and second substrate is about the diameter of the plurality of beads. In some examples, the composition can be removed from the first substrate before analysis of the biological sample by applying heat and melting (e.g., partially or completely) the composition. Additionally, sample detachment can be a problem when performing various analysis methods on a biological sample disposed on a substrate. In some examples, the various methods of preserving biological samples on a substrate as described herein can prevent or minimize sample detachment from the substrate.

Provided herein are methods of preserving a biological sample on a first substrate that include (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, wherein the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the chamber; and (d) assembling the first substrate with a second substrate, thereby preserving the biological sample on the first substrate.

Also, provided herein are methods of preserving a biological sample on a first substrate that include (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, where the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the biological sample within the chamber; (d) analyzing the biological sample on the first substrate; (e) assembling the first substrate with a second substrate, thereby preserving the biological sample on the first substrate; (f) applying heat to the first substrate, thereby melting the spacer agent and removing the second substrate and the spacer agent from the first substrate; (g) contacting the first substrate with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; and (h) performing spatial transcriptomic analysis. Alternatively, after applying heat to the first substrate and melting the spacer agent and removing the second substrate to access the biological sample, that biological sample can be evaluated using other methodologies, reagents, etc. for performing alternative analysis to spatial transcriptomics analysis. For example, the biological sample can be further assayed for markers (e.g., protein, nucleic acids) using reagents for immunohistochemistry, cytology, and/or histology. In some embodiments, the biological sample can be further assayed using cell-based assays, wherein the cell-based assays include, but are not limited to, a cell viability assay, a cell proliferation assay, a cytotoxicity assay, a cell senescence assay, or a cell death assay.

I. Introduction

A preserved tissue, such as that provided by practicing the methods for tissue preservation described herein, can find utility in spatial transcriptomics methods and analysis and other types of analyses described herein. Spatial analysis methodologies and compositions described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods and compositions can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte (e.g., a proxy for an analyte including an analyte capture sequence or a ligation product). For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641, 2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709, 2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322, 2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875, 2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO 2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE 14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020), both of which are available at the 10× Genomics Support Documentation website, and can be used herein in any combination, each of which are incorporated herein by reference in their entireties. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.

Some general terminology that may be used in this disclosure can be found in Section (I)(b) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Typically, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein. Examples of nucleic acid analytes include, but are not limited to, DNA (e.g., genomic DNA, cDNA) and RNA, including coding (e.g., mRNA) and non-coding RNA (e.g., rRNA, tRNA, ncRNA).

A “biological sample” is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In some embodiments, a biological sample can be a tissue section. In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a biological sample (e.g., a fixed and/or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature's relative spatial location within the array. For practicing the methods for tissue preservation in the context of spatial arrays, a biological sample could be placed on a spatially arrayed slide as the first substrate, the biological sample surrounded by the spacer agent, mounting agent added to the created chamber comprising the biological sample, followed by placement of the second substrate. At some point in time (e.g., hours, days, weeks, months later), the second substrate could be removed, the mounting agent removed and the spacer agent melted, thereby allowing for the continued processing of the biological sample for spatial analysis. In some embodiments, spatial analysis includes spatial transcriptomic analysis e.g., spatial capture of mRNA, spatial genomic analysis (e.g., genomic DNA), and/or multiplex analysis (e.g., detecting protein and nucleic acid in a sample with an analyte capture agent as described herein). In some embodiments, spatial transcriptomic analysis is performed on analytes (e.g., nucleic acid) captured directly on the array (e.g., captured by a capture probe on the array). In some embodiments, spatial transcriptomic analysis is performed on analytes (e.g., nucleic acid) by one or more probes that hybridize to a target analyte, where at least one probe includes an analyte capture sequence capable of hybridizing to the capture domain of a capture probe. In such examples, with at least two probes hybridized to a target analyte, the two probes can be ligated to one another (e.g., with a ligase) to generate a ligation product that is then captured by a capture probe on the array.

In some embodiments, once the second substrate is removed, the biological sample mounted on the spatially arrayed slide can be aligned (i.e., sandwiched) with a third substrate that includes a plurality of capture probes.

In some embodiments, the alignment of the spatially arrayed slide and the third substrate is facilitated by a sandwiching process. In some embodiments, the spatially arrayed slide and the third substrate are placed in a substrate holder (e.g., an array alignment device). In some embodiments, the device comprises a sample holder. The device can include an alignment mechanism that aligns the spatially arrayed slide and the third substrate. Thus, the devices can advantageously align the spatially arrayed slide and the third substrate and any samples, barcoded probes, or permeabilization reagents that may be on the surface of the spatially arrayed slide and third substrate. Exemplary devices and exemplary sample holders are described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

In some embodiments, the biological sample and the spatially arrayed slide can be contacted with a reagent medium. In some embodiments, the reagent medium comprises a permeabilization agent. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X100™, Tween-20™, or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N-lauroylsarcosine sodium salt solution). Exemplary permeabilization reagents are described in in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

In some embodiments, the reagent medium comprises a lysis reagent. Lysis solutions can include ionic surfactants such as, for example, sarkosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. Exemplary lysis reagents are described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

In some embodiments, the reagent medium comprises a protease. Exemplary proteases include, e.g., pepsin, trypsin, pepsin, elastase, and proteinase K. Exemplary proteases are described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

In some embodiments, the reagent medium comprises a detergent. Exemplary detergents include sodium dodecyl sulfate (SDS), sarkosyl, saponin, Triton X-100™, and Tween-20™. Exemplary detergents are described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

In some embodiments, the reagent medium comprises a nuclease. In some embodiments, the nuclease comprises an RNase. In some embodiments, the RNase is selected from RNase A, RNase C, RNase H, and RNase I. In some embodiments, the reagent medium comprises one or more of sodium dodecyl sulfate (SDS), proteinase K, pepsin, N-lauroylsarcosine, RNAse, and a sodium salt thereof.

The sample holder is compatible with a variety of different schemes for contacting the aligned portions of the biological sample and array with the reagent medium to promote analyte capture. In some embodiments, the reagent medium is deposited directly on the third substrate (e.g., forming a reagent medium that includes the permeabilization reagent and the feature array), and/or directly on the spatially arrayed slide. In some embodiments, the reagent medium is deposited on the spatially arrayed slide and/or third substrate, and then the spatially arrayed slide and the third substrate aligned in the sandwich configuration such that the reagent medium contacts the aligned portions of the biological sample and array.

In certain embodiments a dried permeabilization reagent is applied or formed as a layer on the spatially arrayed slide or the third substrate or both prior to contacting the sample and the feature array. For example, a reagent can be deposited in solution on the spatially arrayed slide or the third substrate or both and then dried. Drying methods include, but are not limited to spin coating a thin solution of the reagent and then evaporating a solvent included in the reagent or the reagent itself. Alternatively, in other embodiments, the reagent can be applied in dried form directly onto the spatially arrayed slide or the third substrate or both. In some embodiments, the coating process can be done in advance of the analytical workflow and the spatially arrayed slide and the third substrate can be stored pre-coated. Alternatively, the coating process can be done as part of the analytical workflow. In some embodiments, the reagent is a permeabilization reagent. In some embodiments, the reagent is a permeabilization enzyme, a buffer, a detergent, or any combination thereof. In some embodiments, the permeabilization enzyme is pepsin. In some embodiments, the reagent is a dried reagent (e.g., a reagent free from moisture or liquid). In some instances, the substrate that includes the sample (e.g., a histological tissue section) is hydrated. The sample can be hydrated by contacting the sample with a reagent medium, e.g., a buffer that does not include a permeabilization reagent. In some embodiments, the hydration is performed while the spatially arrayed slide and the third substrate are aligned in a sandwich configuration.

In some embodiments, spatial analysis workflows can include a sandwiching process. In some embodiments, the workflow includes provision of the spatially arrayed slide comprising the biological sample. In some embodiments, the workflow includes, mounting the biological sample onto the spatially arrayed slide. In some embodiments wherein the biological sample is a tissue sample, the workflow include sectioning of the tissue sample (e.g., cryostat sectioning). In some embodiments, the workflow includes a fixation step. In some instances, the fixation step can include fixation with methanol. In some instances, the fixation step includes formalin (e.g., 2% formalin).

In some embodiments, the biological sample on the spatially arrayed slide is stained. In some instances, the biological sample is imaged, capturing the stain pattern created during the stain step. In some instances, the biological sample then is destained prior to the sandwiching process.

The biological sample can be stained using known staining techniques, including, without limitation, Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), hematoxylin, Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In some embodiments, the biological sample can be stained using a detectable label (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes) as described elsewhere herein. In some embodiments, a biological sample is stained using only one type of stain or one technique. In some embodiments, staining includes biological staining techniques such as H&E staining. In some embodiments, staining includes biological staining using hematoxylin. In some embodiments, staining includes identifying analytes using fluorescently-conjugated antibodies, e.g., by immunofluorescence. In some embodiments, a biological sample is stained using two or more different types of stains, or two or more different staining techniques. For example, a biological sample can be prepared by staining and imaging using one technique (e.g., H&E staining and brightfield imaging), followed by staining and imaging using another technique (e.g., IHC/IF staining and fluorescence microscopy) for the same biological sample. In some instances, a biological sample on the spatially arrayed slide is stained.

In some instances, methods for immunofluorescence include a blocking step. The blocking step can include the use of blocking probes to decrease unspecific binding of the antibodies. The blocking step can optionally further include contacting the biological sample with a detergent. In some instances, the detergent can include Triton X100™. The method can further include an antibody incubation step. In some embodiments, the antibody incubation step effects selective binding of the antibody to antigens of interest in the biological sample. In some embodiments, the antibody is conjugated to an oligonucleotide (e.g., an oligonucleotide-antibody conjugate as described herein). In some embodiments, the antibody is not conjugated to an oligonucleotide. In some embodiments, the method further comprises an antibody staining step. The antibody staining step can include a direct method of immunostaining in which a labelled antibody binds directly to the analyte being stained for. Alternatively, the antibody staining step can include an indirect method of immunostaining in which a first antibody binds to the analyte being stained for, and a second, labelled antibody binds to the first antibody. In some embodiments, the antibody staining step is performed prior to sandwich assembly. In some embodiments wherein an oligonucleotide-antibody conjugate is used in the antibody incubation step, the method does not comprise an antibody staining step.

In some instances, the methods include imaging the biological sample. In some instances, imaging occurs prior to sandwich assembly. In some instances, imaging occurs while the sandwich configuration is assembled. In some instances, imaging occurs during permeabilization of the biological sample. In some instances, image are captured using high resolution techniques (e.g., having 300 dots per square inch (dpi) or greater). For example, images can be captured using brightfield imaging (e.g., in the setting of hematoxylin or H&E stain), or using fluorescence microscopy to detect adhered labels. In some instances, high resolution images are captured temporally using e.g., confocal microscopy. In some instances, a low resolution image is captured. A low resolution image (e.g., images that are about 72 dpi and normally have an RGB color setting) can be captured at any point of the workflow, including but not limited to staining, destaining, permeabilization, sandwich assembly, and migration of the analytes. In some instances, a low resolution image is taken during permeabilization of the biological sample.

In some embodiments, the locations of the one or more analytes in a biological sample are determined by immunofluorescence. In some embodiments, one or more detectable labels (e.g., fluorophore-labeled antibodies) bind to the one or more analytes that are captured (hybridized to) by a probe on the first slide and the location of the one or more analytes is determined by detecting the labels under suitable conditions. In some embodiments, one or more fluorophore-labeled antibodies are used to conjugate to a moiety that associates with a probe on the first slide or the analyte that is hybridized to the probe on the first slide. In some instances, the location(s) of the one or more analytes is determined by imaging the fluorophore-labeled antibodies when the fluorophores are excited by a light of a suitable wavelength. In some embodiments, the location of the one or more analytes in the biological sample is determined by correlating the immunofluorescence data to an image of the biological sample. In some instances, the tissue is imaged throughout the permeabilization step.

In some instances, the biological samples can be destained. In some instances, destaining occurs prior to permeabilization of the biological sample. By way of example only, H&E staining can be destained by washing the sample in HCl. In some instances, the hematoxylin of the H&E stain is destained by washing the sample in HCl. In some embodiments, destaining can include 1, 2, 3, or more washes in HCl. In some embodiments, destaining can include adding HCl to a downstream solution (e.g., permeabilization solution).

Between any of the methods disclosed herein, the methods can include a wash step (e.g., with SSC (e.g., 0.1×SSC)). Wash steps can be performed once or multiple times (e.g., 1×, 2×, 3×, between steps disclosed herein). In some instances, wash steps are performed for about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds, or about a minute. In some instances, three washes occur for 20 seconds each. In some instances, the wash step occurs before staining the sample, after destaining the sample, before permeabilization the sample, after permeabilization the sample, or any combination thereof.

In some instances, after the sandwiching process the spatially arrayed slide and the third substrate are separated (e.g., such that they are no longer aligned in a sandwich configuration, also referred to herein as opening the sandwich). In some embodiments, subsequent analysis (e.g., cDNA synthesis, library preparation, and sequences) can be performed on the captured analytes after the spatially arrayed slide and the third substrate are separated.

In some embodiments, the process of transferring the ligation product from the spatially arrayed slide to the third substrate is referred to interchangeably herein as a “sandwich process,” “sandwiching process,” or “sandwiching”. The sandwich process is further described in PCT Patent Application Publication No. WO 2020/123320, which is incorporated by reference in its entirety.

A “capture probe” refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain). In some embodiments, a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next-generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some embodiments, more than one analyte type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., protein analytes) can be performed using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and/or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.

In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes). In some cases, capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligations products that serve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3′ or 5′ end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3′ end” indicates additional nucleotides were added to the most 3′ nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3′ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended using reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probes include the sequence of the capture probe and the sequence of the spatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., via DNA sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) act as templates for an amplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and/or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and/or proximity capture, is described in Section (II)(g) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (II)(h) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medical importance. For example, the methods and compositions described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder.

The methods and compositions described herein can also allow for: identification of transcriptome and/or proteome expression profiles (e.g., in healthy and/or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of up- and/or down-regulated genes and/or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in tissue; and identification of genetic variants within tissues (e.g., based on gene and/or protein expression profiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes). As used herein, “contact,” “contacted,” and/or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and/or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). Methods of RTL have been described previously. See, e.g., Credle et al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule). In some instances, the oligonucleotides are DNA molecules. In some instances, one of the oligonucleotides includes at least two ribonucleic acid bases at the 3′ end and/or the other oligonucleotide includes a phosphorylated nucleotide at the 5′ end. In some instances, one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., SplintR ligase) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase (e.g., a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNAse H). The released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample. With respect to the tissue preservation methods disclosed herein, once the spacer agent is melted and removed and the mounting agent is removed, the RTL reagents can be added to the tissue and the associated workflow for spatial analysis using RTL as a workflow can be followed.

During analysis of spatial information, sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain the spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate. For example, specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.

Alternatively, specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array. Where necessary, the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.

When sequence information is obtained for capture probes and/or analytes during analysis of spatial information (e.g., spatial transcriptomics), the locations of the capture probes and/or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See, for example, the Exemplary embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and/or Sample and Array Alignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample. The biological sample can be mounted for example, in a biological sample holder. One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder.

The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and/or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits. The software instructions, when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.

In some cases, the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Application No. 2020/061064 and/or U.S. patent application Ser. No. 16/951,854.

Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and/or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and/or level. Exemplary methods to generate a two- and/or three-dimensional map of the analyte presence and/or level are described in PCT Application No. 2020/053655 and spatial analysis methods are generally described in WO 2020/061108 and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of WO 2020/123320, PCT Application No. 2020/061066, and/or U.S. patent application Ser. No. 16/951,843. Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, to determine a location of a sample or array on a substrate relative to a fiducial marker) and/or for quantitative measurements of sizes and/or distances.

II. Method and Kits for Preserving a Biological Sample

As discussed herein, the ability to preserve biological samples for later analysis is a critical, and often, limiting aspect of biological analysis. While biological samples can be preserved on a substrate (e.g., a slide) for further analysis (e.g., microscopy, spatial analysis, and/or immunohistochemistry), maintaining the integrity (e.g., the morphological characteristics) of the tissue structure, issues such as sample detachment can arise. Thus, described herein are methods of preserving a biological sample using a composition including a mixture where the mixture is a customizable spacer agent including (i) a blocking paste (e.g., grease, PEG, etc.) and (ii) a plurality of beads, between a first substrate and a second substrate (e.g., a cover slide, a membrane, etc.).

In some embodiments, a biological sample can be preserved between a first substrate and a second substrate, wherein a composition is applied around the biological sample, thereby creating a chamber wherein the biological sample can be preserved. In some examples, the gap distance between the first and second substrate is about the diameter of the plurality of beads included in the spacer agent. In some embodiments, the composition applied around the biological sample is customizable. For example, the shape can be customizable (e.g., polygon, irregular) and/or the gap distance (e.g., height) of the spacer agent can be customizable dependent on the size and shape of the beads. As a result, the chamber encompassing the biological sample is also customizable. In some embodiments, a mounting agent (e.g., a saccharide, glycerin, etc.) can be placed in the chamber (e.g., in combination with the biological sample). In some examples, the spacer agent composition can be removed from the first substrate before downstream analysis of the biological sample by applying heat and melting the spacer agent composition. Further, preserving biological samples with a spacer agent and a mounting agent can help prevent detachment of a biological sample from the substrate.

Thus, provided herein are methods of preserving a biological sample, the method including: (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, wherein the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the chamber; and (d) assembling the first substrate with a second substrate, thereby preserving the biological sample on the first substrate.

Also provided herein are methods of preserving a biological sample, the method including: (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, where the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the biological sample within the chamber; (d) analyzing the biological sample on the first substrate; (e) assembling the first substrate with a second substrate, thereby preserving the biological sample on the first substrate; (f) applying heat to the first substrate, thereby melting the spacer agent and removing the second substrate and the spacing agent from the first substrate; (g) contacting the first substrate with an array including a plurality of capture probes, where a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; and (h) performing spatial transcriptomic analysis on the array. Alternatively, the biological sample could be placed on a first substrate wherein the first substrate is already a spatially arrayed slide including capture probes as defined herein.

As used herein, a “substrate” refers to a member with at least one surface that generally functions to provide physical support for biological samples, analytes, and/or any of the other chemical and/or physical moieties, agents, and structures described herein. A substrate functions as a support for direct or indirect attachment of capture probes to features of the array. In addition, in some embodiments, a substrate (e.g., the same substrate or a different substrate) can be used to provide support to a biological sample, particularly, for example, a thin tissue section. Accordingly, a “substrate” is a support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and/or capture probes on the substrate.

Substrates can be formed from a variety of solid materials, gel-based materials, colloidal materials, semi-solid materials (e.g., materials that are at least partially cross-linked), materials that are fully or partially cured, and materials that undergo a phase change or transition to provide physical support. Examples of substrates, including the first, second, and/or third substrate, that can be used in the methods and systems described herein include, but are not limited to, slides (e.g., slides formed from various glasses, slides formed from various polymers), plastics, hydrogels, layers and/or films, membranes (e.g., porous membranes), flow cells, cuvettes, wafers, plates, or combinations thereof. In some embodiments, substrates can optionally include functional elements such as recesses, protruding structures, microfluidic elements (e.g., channels, reservoirs, electrodes, valves, seals), and various markings.

In some embodiments, the first substrate includes a glass surface. In some embodiments, the second substrate includes a glass surface. In some embodiments, the glass surface is a glass slide. In some embodiments, the second substrate includes a porous surface. In some embodiments, the second substrate includes a cellulose membrane. In some embodiments, the second substrate includes a nitrocellulose or nylon membrane. In some embodiments, the second substrate is a coverslip. In some embodiments, the coverslip is plastic. In some embodiments, the plastic coverslip is a flexible plastic coverslip.

In some embodiments, the method includes fixing the biological sample after the first substrate and the second substrate are assembled with the biological sample. For example, the biological sample can be prepared using formalin-fixation and paraffin-embedding (FFPE), which are established methods. In some embodiments, cell suspensions and other non-tissue samples can be prepared using formalin-fixation and paraffin-embedding.

As an alternative to formalin fixation described above, a biological sample can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, a sample can be fixed via immersion in ethanol, methanol, acetone, formaldehyde (e.g., 2% formaldehyde), paraformaldehyde-Triton, glutaraldehyde, or combinations thereof.

In some embodiments, acetone fixation is used with fresh frozen samples, which can include, but are not limited to, cortex tissue, mouse olfactory bulb, human brain tumor, human post-mortem brain, and breast cancer samples. In some embodiments, a compatible fixation method is chosen and/or optimized based on a desired workflow. For example, formaldehyde fixation may be chosen as compatible for workflows using IHC/IF protocols for protein visualization. As another example, methanol fixation may be chosen for workflows emphasizing RNA/DNA library quality. Acetone fixation may be chosen in some applications to permeabilize the tissue. When acetone fixation is performed, pre-permeabilization steps may not be performed. Alternatively, acetone fixation can be performed in conjunction with permeabilization steps. In some embodiments, the biological sample is fixed by heat fixation. In some embodiments, the biological sample is fixed by immersion of the sample tissue in a fixative solution. In some embodiments, the biological sample is fixed by perfusion. In some embodiments, the biological sample is fixed by using a chemical fixative. In some embodiments, the chemical fixative can include formaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, an oxidizing agent (e.g., osmium tetroxide, potassium dichromate, chromic acid, or potassium permanganate), a mercurial (e.g., B-5, Zenker's fixative), a picrate, a Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) fixative, or any combination thereof. In some embodiments, the biological sample is fixed with formaldehyde. In some embodiments, the biological sample is fixed with methanol or acetone.

In some embodiments, the biological sample can be fixed before step (d) of assembling the first substrate with the second substrate. In some embodiments, the biological sample can be fixed after step (d) of assembling the first substrate with the second substrate. In such embodiments, the second substrate is permeable, thereby allowing delivery of fixation reagents through the second substrate.

Spacer Agent

As used herein, a “spacer agent” refers to an agent that separates a first substrate and a second substrate. In some embodiments, the spacer agent is delivered to the first substrate, wherein the spacer agent is applied around a biological sample disposed on the first substrate, thereby generating a chamber around the biological sample. In some embodiments, the biological sample is held between the first and second substrates within the chamber.

In some embodiments, the spacer agent includes a mixture including a blocking paste and a plurality of beads. As used herein, “blocking paste” refers to a composition (e.g., a composition such as a paste, a gel, a cement, etc.) that can be either hydrophobic or hydrophilic. In some embodiments, the blocking paste includes a hydrophilic agent. In some embodiments, the hydrophilic agent includes hydrophilic polymer(s) and/or hydrophilic nanoparticles. In some embodiments, the blocking paste includes a hydrophobic agent. In some embodiments, the hydrophobic agent can include a grease. In some embodiments, the grease includes one or more of a vacuum grease, an apiezon grease, a lithium grease, a silicone grease, a Fomblin grease, and a TLC grease. In some embodiments, the method includes partially or completely melting the grease and removing the second substrate from the first substrate. In some embodiments, the grease can be melted and removed by applying heat to the substrate. In some embodiments, the grease has a melting temperature of about 25° C. to about 250° C. (e.g., about 25° C. to about 225° C., about 25° C. to about 200° C., about 25° C. to about 175° C., about 25° C. to about 150° C., about 25° C. to about 125° C., about 25° C. to about 100° C., about 25° C. to about 75° C., about 25° C. to about 50° C., about 50° C. to about 250° C., about 50° C. to about 225° C., about 50° C. to about 200° C., about 50° C. to about 175° C., about 50° C. to about 150° C., about 50° C. to about 125° C., about 50° C. to about 100° C., about 50° C. to about 75° C., about 75° C. to about 250° C., about 75° C. to about 225° C., about 75° C. to about 200° C., about 75° C. to about 175° C., about 75° C. to about 150° C., about 75° C. to about 125° C., about 75° C. to about 100° C., about 100° C. to about 250° C., about 100° C. to about 225° C., about 100° C. to about 200° C., about 100° C. to about 175° C., about 100° C. to about 150° C., about 100° C. to about 125° C., about 125° C. to about 250° C., about 125° C. to about 225° C., about 125° C. to about 200° C., about 125° C. to about 175° C., about 125° C. to about 150° C., about 150° C. to about 250° C., about 150° C. to about 225° C., about 150° C. to about 200° C., about 150° C. to about 175° C., about 175° C. to about 250° C., about 175° C. to about 225° C., about 175° C. to about 200° C., about 200° C. to about 250° C., about 200° C. to about 225° C., or about 225° C. to about 250° C.). In some embodiments, the grease has a melting temperature of about 30° C. In some embodiments, the grease has a melting temperature of about 90° C. to about 150° C. (e.g., about 90° C. to about 140° C., about 90° C. to about 130° C., about 90° C. to about 120° C., about 90° C. to about 110° C., about 90° C. to about 100° C., about 100° C. to about 150° C., about 100° C. to about 140° C., about 100° C. to about 130° C., about 100° C. to about 120° C., about 100° C. to about 110° C., about 110° C. to about 150° C., about 110° C. to about 140° C., about 110° C. to about 130° C., about 110° C. to about 120° C., about 120° C. to about 150° C., about 120° C. to about 140° C., about 120° C. to about 130° C., about 130° C. to about 150° C., about 130° C. to about 140° C., or about 140° C. to about 150° C.). In some embodiments, the grease has a melting temperature of about 100° C. In some embodiments, the grease has a melting temperature that is higher than about 25° C. In some embodiments, the grease has a melting temperature that is higher than about 100° C.

In some embodiments, the hydrophobic agent is polyethylene glycol (PEG). In some embodiments, the PEG has a molecular weight of about 400 daltons to about 6,000 daltons (e.g., about 400 to about 5,500, about 400 to about 5,000, about 400 to about 4,500, about 400 to about 4,000, about 400 to about 3,500, about 400 to about 3,000, about 400 to about 2,500, about 400 to about 2,000, about 400 to about 1,500, about 400 to about 1,000, about 400 to about 500, about 500 to about 6,000, about 500 to about 5,500, about 500 to about 5,000, about 500 to about 4,500, about 500 to about 4,000, about 500 to about 3,500, about 500 to about 3,000, about 500 to about 2,500, about 500 to about 2,000, about 500 to about 1,500, about 500 to about 1,000, about 1,000 to about 6,000, about 1,000 to about 5,500, about 1,000 to about 5,000, about 1,000 to about 4,500, about 1,000 to about 4,000, about 1,000 to about 3,500, about 1,000 to about 3,000, about 1,000 to about 2,500, about 1,000 to about 2,000, about 1,000 to about 1,500, about 1,500 to about 6,000, about 1,500 to about 5,500, about 1,500 to about 5,000, about 1,500 to about 4,500, about 1,500 to about 4,000, about 1,500 to about 3,500, about 1,500 to about 3,000, about 1,500 to about 2,500, about 1,500 to about 2,000, about 2,000 to about 6,000, about 2,000 to about 5,500, about 2,000 to about 5,000, about 2,000 to about 4,500, about 2,000 to about 4,000, about 2,000 to about 3,500, about 2,000 to about 3,000, about 2,000 to about 2,500, about 2,500 to about 6,000, about 2,500 to about 5,500, about 2,500 to about 5,000, about 2,500 to about 4,500, about 2,500 to about 4,000, about 2,500 to about 3,500, about 2,500 to about 3,000, about 3,000 to about 6,000, about 3,000 to about 5,500, about 3,000 to about 5,000, about 3,000 to about 4,500, about 3,000 to about 4,000, about 3,000 to about 3,500, about 3,500 to about 6,000, about 3,500 to about 5,500, about 3,500 to about 5,000, about 3,500 to about 4,500, about 3,500 to about 4,000, about 4,000 to about 6,000, about 4,000 to about 5,500, about 4,000 to about 5,000, about 4,000 to about 4,500, about 4,500 to about 6,000, about 4,500 to about 5,500, about 4,500 to about 5,000, about 5,000 to about 6,000, about 5,000 to about 5,500, or about 5,500 to about 6,000). In some embodiments, the PEG has a molecular weight of about 500 daltons. In some embodiments, the PEG has a molecular weight of about 1,000 daltons. In some embodiments, the PEG has a molecular weight of about 3,000 daltons. In some embodiments, the PEG has a molecular weight of about 4,500 daltons. In some embodiments, the PEG has a molecular weight of about 6,000 daltons.

In some embodiments, the PEG has a melting temperature between about 25° C. and 250° C. (e.g., about 25° C. to about 225° C., about 25° C. to about 200° C., about 25° C. to about 175° C., about 25° C. to about 150° C., about 25° C. to about 125° C., about 25° C. to about 100° C., about 25° C. to about 75° C., about 25° C. to about 50° C., about 50° C. to about 250° C., about 50° C. to about 225° C., about 50° C. to about 200° C., about 50° C. to about 175° C., about 50° C. to about 150° C., about 50° C. to about 125° C., about 50° C. to about 100° C., about 50° C. to about 75° C., about 75° C. to about 250° C., about 75° C. to about 225° C., about 75° C. to about 200° C., about 75° C. to about 175° C., about 75° C. to about 150° C., about 75° C. to about 125° C., about 75° C. to about 100° C., about 100° C. to about 250° C., about 100° C. to about 225° C., about 100° C. to about 200° C., about 100° C. to about 175° C., about 100° C. to about 150° C., about 100° C. to about 125° C., about 125° C. to about 250° C., about 125° C. to about 225° C., about 125° C. to about 200° C., about 125° C. to about 175° C., about 125° C. to about 150° C., about 150° C. to about 250° C., about 150° C. to about 225° C., about 150° C. to about 200° C., about 150° C. to about 175° C., about 175° C. to about 250° C., about 175° C. to about 225° C., about 175° C. to about 200° C., about 200° C. to about 250° C., about 200° C. to about 225° C., or about 225° C. to about 250° C.).

In some embodiments, the plurality of beads includes a single type of bead (e.g., approximately uniform in volume, shape, and other physical properties, such as translucence). In some embodiments, the plurality of beads includes two or more types of different beads.

In some embodiments, the beads in the plurality of beads have a diameter from about 5 μm to about 80 μm (e.g., about 5 μm to about 70 μm, about 5 μm to about 60 μm, about 5 μm to about 50 μm, about 5 μm to about 40 μm, about 5 μm to about 30 μm, about 5 μm to about 20 μm, about 5 μm to about 10 μm, about 10 μm to about 80 μm, about 10 μm to about 70 μm, about 10 μm to about 60 μm, about 10 μm to about 50 μm, about 10 μm to about 40 μm, about 10 μm to about 30 μm, about 10 μm to about 20 μm, about 20 μm to about 80 μm, about 20 μm to about 70 μm, about 20 μm to about 60 μm, about 20 μm to about 50 μm, about 20 μm to about 40 μm, about 20 μm to about 30 μm, about 30 μm to about 80 μm, about 30 μm to about 70 μm, about 30 μm to about 60 μm, about 30 μm to about 50 μm, about 30 μm to about 40 μm, about 40 μm to about 80 μm, about 40 μm to about 70 μm, about 40 μm to about 60 μm, about 40 μm to about 50 μm, about 50 μm to about 80 μm, about 50 μm to about 70 μm, about 50 μm to about 60 μm, about 60 μm to about 80 μm, about 60 μm to about 70 μm, or about 70 μm to about 80 μm). In some embodiments, the plurality of beads (e.g., a bead in the plurality of beads) has a diameter of about 5 μm. In some embodiments, the plurality of beads has a diameter of about 10 μm. In some embodiments, the plurality of beads has a diameter of about 15 μm. In some embodiments, the plurality of beads has a diameter of about 20 μm. In some embodiments, the plurality of beads has a diameter of about 25 μm. In some embodiments, the plurality of beads has a diameter of about 30 μm. In some embodiments, the plurality of beads has a diameter of about 35 μm. In some embodiments, the plurality of beads has a diameter of about 40 μm.

In some embodiments, the spacer agent generates a gap distance between the first substrate and the second substrate, wherein the gap distance (e.g., height of the chamber) is about the diameter of the plurality of beads.

In some embodiments, the spacer agent can be produced by mixing a blocking paste and a plurality of beads. In some embodiments, the spacer agent includes a mixture of: (i) the blocking paste, and (ii) the plurality of beads, wherein the mixture is from about 1×10⁸ beads/mL in about 0.5 cm³ of the blocking paste to about 1×10¹² beads/mL in about 2 cm³ of the blocking paste (e.g., a hydrophobic agent or hydrophilic agent). In some embodiments, the spacer agent comprises a mixture of: (i) the blocking paste, and (ii) the plurality of beads, wherein the mixture is from about 1×10⁹ beads/mL in about 1.0 cm³ of the blocking paste to about 1×10¹¹ beads/mL in about 2 cm³ of the blocking paste. In some embodiments, the spacer agent comprises a mixture of: (i) the blocking paste, and (ii) the plurality of beads, wherein the mixture is from about 1×10¹⁰ beads/mL in about 1.0 cm³ of the blocking paste to about 1×10¹¹ beads/mL in about 1.5 cm³ of the blocking paste.

Mounting Agent

As used herein, a “mounting agent” refers to an agent that is used to adhere a second substrate (e.g., a cover slide, a membrane, etc.) to the first substrate (e.g., a glass slide) and to preserve the biological sample during, for example, handling, and/or storage. In some embodiments, the mounting agent can cover the biological sample within the chamber generated by the spacer agent. In some embodiments, the mounting agent can be held between the first and second substrates and within the chamber.

In some embodiments, the mounting agent can include aqueous glycerol, lactophenol-based fluid media, water-soluble mounting media (e.g., glycerol-gelatin, gum-chloral), Vectashield, CyGEL, or any combinations thereof. In some embodiments, the mounting agent is glycerin. In some embodiments, the mounting agent is a carbohydrate. In some embodiments, the mounting agent is a saccharide. In some embodiments, the saccharide is a monosaccharide. In some embodiments, the saccharide is a disaccharide. In some embodiments, the saccharide is sucrose. In some embodiments, the saccharide is polysucrose.

In some embodiments, the mounting agent preserves the biological sample (e.g., a tissue section) in an environment suitable for preservation. In some embodiments, preservation includes, but is not limited to hydrating the biological sample. In such embodiments, removing the second substrate after a period of time is performed without damaging the biological sample. In some embodiments, physical properties of the mounting agent affect the imaging the biological sample. For example, the viscosity and the refractive index of the mounting agent can affect imaging the biological sample. In some embodiments, less viscous mounting agents can be used in the preservation of a biological sample in any of the methods described herein. For example, saccharides (e.g., sucrose) are generally less viscous than glycerin. In some embodiments, a mounting agent that has about the same refractive index of the second substrate is suitable for microscopy methods. For example, if the mounting agent has about the same refractive index as the second substrate, it provides better conditions for microscopy (e.g., imaging) than if the mounting agent does not have a refractive index of about the same as the second substrate. Thus, in some embodiments, the mounting agent, (e.g., sucrose) has about the same refractive index as the second substrate. In some embodiments, when the mounting agent has about the same refractive index as the second substrate, microscopy methods, including, but not limited to, fluorescent microscopy and/or bright field microscopy, result in improved imaging as compared to when the mounting agent does not have about the same refractive index of the second substrate.

In some embodiments, the mounting agent can be used to preserve a biological sample. In some embodiments, the biological sample can include a tissue sample, a tissue section, an organ, an organism, or a cell culture sample. In some embodiments, the biological sample can be preserved for up to 1 month (e.g., up to 1 week, up to 2 weeks, or up to 3 weeks). In some embodiments, the biological sample can be preserved for up to 12 months or more (e.g., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months).

In some embodiments, the methods provided herein include imaging the biological sample. In some embodiments, the biological sample can be imaged using brightfield imaging or fluorescence microscopy. In some embodiments, the method can include: (i) separating the first substrate from the second substrate; (ii) contacting the first substrate with an array including a plurality of capture probes, where a capture probe of the plurality capture probes comprises: (i) a spatial barcode and (ii) a capture domain; and (iii) performing spatial transcriptomic analysis (as described herein). In some embodiments, the spatial transcriptomic analysis can include sequencing.

In some embodiments, the separating step includes melting (e.g., partially or completely) a spacer agent between the first substrate and the second substrate and removing the second substrate from the first substrate.

Composition and Kits for Preserving a Biological Sample

Also provided herein are compositions for the preserving a biological sample (e.g., on a first substrate), including (i) a blocking paste; and (ii) a plurality of beads.

In some compositions the blocking paste includes a hydrophilic agent (e.g., any of the hydrophilic agents described herein). In some embodiments, the blocking paste includes a hydrophobic agent. In some embodiments, the hydrophobic agent is a grease. For example, the grease can include one or more of a vacuum grease, an apiezon grease, a lithium grease, a silicone grease, a Fomblin grease, or a TLC grease as previously described.

In some embodiments, the grease has a melting temperature of about 25° C. to about 250° C. and any value in between. In some embodiments, the grease has a melting temperature of about 30° C. In some embodiments, the grease has a melting temperature of about 90° C. to about 150° C. In some embodiments, the grease has a melting temperature of about 100° C. In some embodiments, the grease has a melting temperature that is higher than 25° C. In some embodiments, the grease has a melting temperature that is higher than 100° C.

In some embodiments, the hydrophobic agent includes polyethylene glycol (PEG). In some embodiments, the PEG has a molecular weight of about 400 daltons to about 6,000 daltons, or any value in between. In some embodiments, the PEG has a molecular weight of about 500 daltons. In some embodiments, the PEG has a molecular weight of about 1,000 daltons. In some embodiments, the PEG has a molecular weight of about 3,000 daltons. In some embodiments, the PEG has a molecular weight of about 4,500 daltons. In some embodiments, the PEG has a molecular weight of about 6,000 daltons.

In some embodiments, the PEG has a melting temperature between about 25° C. and 250° C.

In some embodiments, a bead of the plurality of beads includes a diameter from about 5 μm to about 80 μm, or any value in between. In some embodiments, a bead of the plurality of beads has a diameter of about 5 μm. In some embodiments, a bead of the plurality of beads has a diameter of about 10 μm. In some embodiments, a bead of the plurality of beads has a diameter of about 20 μm. In some embodiments, a bead of the plurality of beads has a diameter of about 30 μm. In some embodiments, a bead of the plurality of beads has a diameter of about 40 μm.

In some embodiments, the composition includes a mixture of: (i) the blocking paste, and (ii) the plurality of beads (e.g., any of the beads described herein), wherein the mixture is from about 1×10⁸ beads/mL in about 0.5 cm³ of the blocking paste to about 1×10¹² beads/mL in about 2 cm³ of the blocking paste.

In some embodiments, the composition includes a mixture of: (i) the blocking paste, and (ii) the plurality of beads, wherein the mixture is from about 1×10⁹ beads/mL in about 1.0 cm³ of the blocking paste to about 1×10¹¹ beads/mL in about 2 cm′ of the blocking paste.

In some embodiments, the composition includes a mixture of: (i) the blocking paste, and (ii) the plurality of beads, wherein the mixture is from about 1×10¹⁰ beads/mL in about 1.0 cm³ of the blocking paste to about 1×10¹¹ beads/mL in about 1.5 cm³ of the blocking paste.

Also provided herein are kits including (a) a spacer agent; and (b) a mounting agent. In some kits, the spacer agent includes a mixture including a blocking paste and a plurality of beads. In some kits, the blocking paste incudes a hydrophilic agent (e.g., any of the hydrophilic agents described herein). In some embodiments, the blocking paste includes a hydrophobic agent. In some kits, the hydrophobic agent includes a grease. Non-limiting examples of greases include one or more of a vacuum grease, an apiezon grease, a lithium grease, a silicone grease, a Fomblin grease, and a TLC grease.

In some kits, the grease has a melting temperature of about 25° C. to about 250° C. In some kits, the grease has a melting temperature of about 30° C. In some kits, the grease has a melting temperature of about 90° C. to about 150° C. In some kits, the grease has a melting temperature of about 100° C. In some kits, the grease has a melting temperature that is higher than about 25° C. In some kits, the grease has a melting temperature that is higher than about 100° C.

In some kits, the hydrophobic agent includes polyethylene glycol (PEG). In some kits, the PEG has a molecular weight of about 400 daltons to about 6,000 daltons (e.g., any of the molecular weights described herein). In some kits, the PEG has a molecular weight of about 500 daltons. In some kits, the PEG has a molecular weight of about 1,000 daltons. In some kits, the PEG has a molecular weight of about 3,000 daltons. In some kits, the PEG has a molecular weight of about 4,500 daltons. In some kits, the PEG has a molecular weight of about 6,000 daltons.

In some kits, the PEG has a melting temperature between about 25° C. and 250° C. and anywhere in between. In some kits, a bead of the plurality of beads has a diameter (e.g., any of the diameters described herein) from about 5 μm to about 80 μm. In some kits, a bead of the plurality of beads has a diameter of about 5 μm. In some kits, a bead of the plurality of beads has a diameter of about 10 μm. In some kits, a bead of the plurality of beads has a diameter of about 20 μm. In some kits, a bead of the plurality of beads has a diameter of about 30 μm. In some kits, a bead of the plurality of beads has a diameter of about 40 μm.

In some kits, the spacer agent includes a mixture of: (i) the blocking paste, and (ii) the plurality of beads, wherein the mixture is from about 1×10⁸ beads/mL in about 0.5 cm³ of the blocking paste to about 1×10¹² beads/mL in about 2 cm³ of the blocking paste.

In some kits, the spacer agent includes a mixture of: (i) the blocking paste, and (ii) the plurality of beads, wherein the mixture is from about 1×10⁹ beads/mL in about 1.0 cm³ of the blocking paste to about 1×10¹¹ beads/mL in about 2 cm³ of the blocking paste.

In some kits, the spacer agent includes a mixture of: (i) the blocking paste, and (ii) the plurality of beads, wherein the mixture is from about 1×10¹⁰ beads/mL in about 1.0 cm³ of the blocking paste to about 1×10¹¹ beads/mL in about 1.5 cm³ of the blocking paste.

In some kits, the mounting agent includes glycerin. In some kits, the mounting agent includes a saccharide. In some kits, the saccharide includes sucrose.

EXAMPLES Example 1—Methods for Preserving a Biological Sample

FIGS. 1A-1B show an exemplary configuration of a first substrate, a spacer agent, a mounting agent, and a second substrate of the methods, compositions, and kits described herein. FIG. 1A shows a first substrate (e.g., a glass slide) where a spacer agent including: (i) a blocking paste and (ii) a plurality of beads is applied to the first substrate. The spacer agent generates a chamber where a mounting agent is applied and retained by the spacer agent. In some embodiments, a biological sample is disposed on the first substrate before or after the spacer agent is disposed on the first substrate. In some embodiments, the biological sample is disposed on the first substrate before the mounting agent. A second substrate can then be disposed above the first substrate thereby sealing the mounting agent, and optionally, a biological sample in the chamber generated by the spacer agent. In embodiments that include a biological sample, the biological sample can be preserved for analysis, including spatial transcriptomic analysis, at a later time (e.g., 2, 3, 4, 5, 6 months, or more) after preservation. FIG. 1B shows a side view of the exemplary configuration shown in FIG. 1A.

In a non-limiting example, a biological sample is disposed on a first substrate (e.g., a glass slide). A spacer agent is then applied to a glass slide around the biological sample, thereby generating a chamber around the biological sample. In some examples, the blocking paste includes a hydrophilic agent (e.g., any of the hydrophilic agents described herein). In some examples, the blocking paste includes a hydrophobic agent. In some examples, the hydrophobic agent includes a vacuum grease, apiezon grease, or polyethylene glycol and is applied to the glass slide in a customizable shape (see, e.g., FIGS. 2A-2B).

A mounting agent is delivered to the biological sample within the chamber, where the mounting agent covers the biological sample. The glass slide (e.g., a first substrate) is assembled with a cover slip (e.g., a second substrate) completely sealing the biological sample in the chamber, thereby allowing the sample to be preserved and stored for up to several months. The second substrate is assembled with the first substrate to seal the chamber in any orientation, such as in a parallel orientation, perpendicular orientation, or any orientation therein between. The spacer agent can include a blocking paste (e.g., a hydrophilic agent or a hydrophobic agent) and a plurality of beads, wherein once the glass slide and cover slip are assembled, the spacer agent provides a gap distance (e.g., a height) between the glass slide and the cover slip where the gap distance is about the diameter of the plurality of beads. For example, the gap distance can be approximately 10 μm when the plurality of beads are polystyrene beads with about a 10 μm diameter (FIG. 3 ). The gap distance can be approximately 20 μm when the plurality of beads are polystyrene beads with about a 20 μm diameter (FIG. 4 ). The gap distance can be approximately 30 μm when the plurality of beads are polystyrene beads with about a 30 μm diameter (FIG. 5 ). The gap distance can be approximately 40 μm when the plurality of beads are polystyrene beads with about a 40 μm diameter (FIG. 6 ). It is contemplated that the blocking paste will also influence the gap distance to some extent, however not to the extent of the beads with a known diameter. Furthermore, the gap distance can be customized according to the thickness of the biological sample, wherein different greases and different beads (e.g., beads with a different diameter) can be used in the spacer agent and the spacer agent can be applied to the glass slide in various shapes (see e.g., FIGS. 2A and 2B). In some examples, a flexible plastic cover slip (e.g., porous or mesaporous plastic membrane) can be used to fix the biological sample after assembling the glass slide and cover slip (FIG. 7 ) and the cover slip can be removed to recover the biological sample in the chamber. 

What is claimed is:
 1. A method of preserving a biological sample on a first substrate, the method comprising: (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, wherein the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the chamber; and (d) assembling the first substrate with a second substrate, thereby preserving the biological sample on the first substrate.
 2. The method of claim 1, wherein the first substrate and/or the second substrate comprises a glass slide.
 3. The method of claim 1, wherein the first substrate comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain.
 4. The method of claim 1, wherein the second substrate comprises a porous surface, wherein the porous surface comprises a cellulose membrane.
 5. The method of claim 4, wherein the method further comprises fixing the biological sample after the first substrate and the second substrate are assembled with the biological sample.
 6. The method of claim 5, wherein the biological sample is fixed with formaldehyde, methanol, or acetone.
 7. The method of claim 1, wherein the spacer agent comprises a mixture comprising a blocking paste and a plurality of beads.
 8. The method of claim 7, wherein the mixture comprises from about 1×10⁸ beads/mL in about 0.5 cm³ of the blocking paste to about 1×10¹² beads/mL in about 2 cm³ of the blocking paste.
 9. The method of claim 7, wherein the blocking paste comprises a hydrophobic agent.
 10. The method of claim 9, wherein the hydrophobic agent comprises a grease, wherein the grease comprises one or more of a vacuum grease, an apiezon grease, a lithium grease, a silicone grease, a Fomblin grease, and a TLC grease.
 11. The method of claim 10, wherein the method further comprises partially or completely melting the grease and removing the second substrate from the first substrate.
 12. The method of claim 11, wherein the grease has a melting temperature of about 25° C. to about 250° C.
 13. The method of claim 9, wherein the hydrophobic agent comprises polyethylene glycol (PEG), wherein the PEG has a molecular weight of about 400 daltons to about 6,000 daltons, and wherein the PEG has a melting temperature between about 25° C. and 250° C.
 14. The method of claim 7, wherein a bead in the plurality of beads has a diameter from about 5 μm to about 80 μm.
 15. The method of claim 14, wherein the spacer agent generates a gap distance between the first substrate and the second substrate, wherein the gap distance is about the diameter of the bead.
 16. The method of claim 1, wherein the mounting agent comprises glycerin or a saccharide.
 17. The method of claim 1, wherein the biological sample comprises a tissue sample, a tissue section, an organ, an organism, or a cell culture sample.
 18. The method of claim 1, wherein the method further comprises imaging the biological sample.
 19. The method of claim 1, wherein the method further comprises: (i) separating the first substrate from the second substrate; (ii) contacting the first substrate with an array comprising a plurality of capture probes, wherein a capture probe of the plurality capture probes comprises: (i) a spatial barcode and (ii) a capture domain; and (iii) performing spatial transcriptomic analysis.
 20. The method of claim 19, wherein the spatial transcriptomic analysis comprises sequencing.
 21. A method of preserving and analyzing a biological sample, the method comprising: (a) disposing a biological sample on a first substrate; (b) delivering a spacer agent to the first substrate, wherein the spacer agent is applied around the biological sample on the first substrate, thereby generating a chamber around the biological sample; (c) delivering a mounting agent to the biological sample within the chamber; (d) analyzing the biological sample on the first substrate; (e) assembling the first substrate with a second substrate such that the chamber is sealed, thereby preserving the biological sample on the first substrate; (f) applying heat to the first substrate, thereby partially or completely melting the spacer agent and removing the second substrate and the spacer agent from the first substrate; (g) contacting the first substrate with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; and (h) performing spatial transcriptomic analysis. 